Rate Of Clonal Expansion Research Articles

Evolution of myeloproliferative neoplasms from normal blood stem cells.

Over the course of the last decade, genomic studies in the context of normal human hematopoiesis have provided new insights into the early pathogenesis of myeloproliferative neoplasms (MPN). A preclinical phase of MPN, termed clonal hematopoiesis (CH) was identified and subsequent lineage tracing studies revealed a multi-decade long time interval from acquisition of an MPN phenotypic driver mutation in a hematopoietic stem cell (HSC) to the development of overt MPN. Multiple germline variants associated with MPN risk have been identified through genome-wide association studies (GWAS) and in some cases functional interrogation of the impact of the variant has uncovered new insights into HSC biology and MPN development. Increasingly sophisticated methods to study clonal contributions to human hematopoiesis and measure HSC fitness have helped discern the biology underlying the tremendous clinical heterogeneity observed in MPN. Despite these advances, significant knowledge gaps remain particularly with respect to germline genetic contributors to both MPN pathogenesis and phenotypic diversity, as well as limitations in the ability to prospectively quantify rates of clonal expansion in individual MPN patients. Ultimately, we envisage a personalized approach to MPN care in the future, where an individualized genetic assessment can predict MPN trajectory and this information will be used to inform and guide therapy. MPN is particularly amenable to precision medicine strategies and our increased understanding of the evolution of MPN from normal blood stem cells provides a unique opportunity for early therapeutic intervention approaches and potentially MPN prevention strategies.

Epigenetic and proteomic signatures associate with clonal hematopoiesis expansion rate.

Clonal hematopoiesis of indeterminate potential (CHIP), whereby somatic mutations in hematopoietic stem cells confer a selective advantage and drive clonal expansion, not only correlates with age but also confers increased risk of morbidity and mortality. Here, we leverage genetically predicted traits to identify factors that determine CHIP clonal expansion rate. We used the passenger-approximated clonal expansion rate method to quantify the clonal expansion rate for 4,370 individuals in the National Heart, Lung, and Blood Institute (NHLBI) Trans-Omics for Precision Medicine (TOPMed) cohort and calculated polygenic risk scores for DNA methylation aging, inflammation-related measures and circulating protein levels. Clonal expansion rate was significantly associated with both genetically predicted and measured epigenetic clocks. No associations were identified with inflammation-related lab values or diseases and CHIP expansion rate overall. A proteome-wide search identified predicted circulating levels of myeloid zinc finger 1 and anti-Müllerian hormone as associated with an increased CHIP clonal expansion rate and tissue inhibitor of metalloproteinase 1 and glycine N-methyltransferase as associated with decreased CHIP clonal expansion rate. Together, our findings identify epigenetic and proteomic patterns associated with the rate of hematopoietic clonal expansion.

Determinants of mosaic chromosomal alteration fitness

Clonal hematopoiesis (CH) is characterized by the acquisition of a somatic mutation in a hematopoietic stem cell that results in a clonal expansion. These driver mutations can be single nucleotide variants in cancer driver genes or larger structural rearrangements called mosaic chromosomal alterations (mCAs). The factors that influence the variations in mCA fitness and ultimately result in different clonal expansion rates are not well understood. We used the Passenger-Approximated Clonal Expansion Rate (PACER) method to estimate clonal expansion rate as PACER scores for 6,381 individuals in the NHLBI TOPMed cohort with gain, loss, and copy-neutral loss of heterozygosity mCAs. Our mCA fitness estimates, derived by aggregating per-individual PACER scores, were correlated (R2 = 0.49) with an alternative approach that estimated fitness of mCAs in the UK Biobank using population-level distributions of clonal fraction. Among individuals with JAK2 V617F clonal hematopoiesis of indeterminate potential or mCAs affecting the JAK2 gene on chromosome 9, PACER score was strongly correlated with erythrocyte count. In a cross-sectional analysis, genome-wide association study of estimates of mCA expansion rate identified a TCL1A locus variant associated with mCA clonal expansion rate, with suggestive variants in NRIP1 and TERT.

Dynamics of clonal hematopoiesis under DNA-damaging treatment in patients with ovarian cancer

Clonal hematopoiesis (CH) driven by mutations in the DNA damage response (DDR) pathway is frequent in patients with cancer and is associated with a higher risk of therapy-related myeloid neoplasms (t-MNs). Here, we analyzed 423 serial whole blood and plasma samples from 103 patients with relapsed high-grade ovarian cancer receiving carboplatin, poly(ADP-ribose) polymerase inhibitor (PARPi) and heat shock protein 90 inhibitor (HSP90i) treatment within the phase II EUDARIO trial using error-corrected sequencing of 72 genes. DDR-driven CH was detected in 35% of patients and was associated with longer duration of prior PARPi treatment. TP53- and PPM1D-mutated clones exhibited substantially higher clonal expansion rates than DNMT3A- or TET2-mutated clones during treatment. Expansion of DDR clones correlated with HSP90i exposure across the three study arms and was partially abrogated by the presence of germline mutations related to homologous recombination deficiency. Single-cell DNA sequencing of selected samples revealed clonal exclusivity of DDR mutations, and identified DDR-mutated clones as the origin of t-MN in two investigated cases. Together, these results provide unique insights into the architecture and the preferential selection of DDR-mutated hematopoietic clones under intense DNA-damaging treatment. Specifically, PARPi and HSP90i therapies pose an independent risk for the expansion of DDR-CH in a dose-dependent manner.

Cost-Effective and Scalable Clonal Hematopoiesis Assay Provides Insight into Clonal Dynamics

Clonal hematopoiesis of indeterminate potential (CHIP) is a common age-related phenomenon that occurs when hematopoietic stem cells acquire mutations in a select set of genes commonly mutated in myeloid neoplasia which then expand clonally. Current sequencing assays to detect CHIP mutations are not optimized for the detection of these variants and can be cost-prohibitive when applied to large cohorts or serial sequencing. In this study, an affordable (∼$8 per sample), accurate, and scalable sequencing assay for CHIP is introduced and validated. The efficacy of the assay is demonstrated by identifying CHIP mutations in a cohort of 456 individuals with DNA collected at multiple timepoints in the Vanderbilt BioVU biobank and quantifying clonal expansion rates over time. A total of 101 individuals with CHIP/CCUS were identified, and individual-level clonal expansion rate was calculated using the variant allele fraction (VAF) at both timepoints. Differences in clonal expansion rate by driver gene were observed, but there was also significant individual-level heterogeneity, emphasizing the multifactorial nature of clonal expansion. Additionally, the study explores mutation co-occurrence and clonal competition between multiple driver mutations.

Abstract 6929: Inter- and intratumoral PIK3CA subclonal diversity in breast cancer contextualized by single-cell multiomics

Abstract Rare clonotypes within pre-cancerous tissues can drive progression to cancer. However, the evolution of rare clonotypes in tumors or normal tissue cannot be defined in the absence of single-cell resolution. At this single-cell level, multiomic interrogation across the Central Dogma of Biology provides enhanced power to reconstruct such evolutionary trajectories, defining the mutational profile, cell identify, and receptor expression within each subpopulation. Leveraging a multiomic approach, we aimed to define how different mutations in the same oncogenic driver observed in the same tumor resection associate with copy number variation (CNV) across the genome. We analyzed individual ductal carcinoma in situ/invasive ductal carcinoma cells using a unified whole-genome and full-transcript RNAseq workflow (ResolveOME™, BioSkryb Genomics) coupled with panel-level extracellular protein information through oligo-conjugated antibodies (BioLegend). We sequenced the exomes and transcriptomes of ResolveOME-amplified single cells from mastectomy samples from twelve patients. At the single nucleotide variant (SNV) level, we identified an allelic series of PIK3CA oncogenic driver mutations in the same tumor resection. A single amino acid, in-frame deletion of E109 dominated the sample, followed by H1047R and K111E in decreasing subclonal abundance. A fourth mutation, E345T, not present in the first sample, was detected as the sole PIK3CA variant in the second tumor sample. Intriguingly, each respective PIK3CA mutation class was associated with a distinct copy number alteration profile revealed by low-coverage whole-genome sequencing: Cells harboring the predominant ΔE109 mutation displayed chromosome 8p,16p, and 17 loss while the less abundant H1047R mutation was in single cells harboring 1q gain, 4q loss, and 22 loss in addition to the 8p and 16q loss present in the ΔE109 cells. PIK3CA K111E had a quiescent, 2n copy number profile. The transcriptomic arm of ResolveOME, containing an oligo-conjugated antibody readout of surface protein expression, jointly confirmed the epithelial identity for the cells harboring the oncogenic PIK3CA mutations. A subpopulation of cells harboring prototypical breast cancer CNV were typed as non-epithelial with increased stemness characteristics, indicative of the ability to resolve phenotypic cellular states. These results suggest a tight interrelationship between CNV and SNV influencing the relative rate of clonal expansion. They also provide the opportunity to explore CNV:SNV signature association with loci exclusive of PIK3CA, and to exploit the power of multiomic integration for lineage reconstruction and for defining common oncogenic signatures of the evolving tumors. Citation Format: Jon Zawistowski, Isai Salas-Gonzalez, Tia Tate, Tatiana Morozova, Katherine Kennedy, Durga Arvapalli, Jamie Remington, Jeffrey Marks, E. Shelley Hwang, Gary Harton, Victor Weigman, Jay A. West. Inter- and intratumoral PIK3CA subclonal diversity in breast cancer contextualized by single-cell multiomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6929.

Inflammatory Signaling Mediated By Gasdermin D Underlies Tet2-Mutant Clonal Hematopoiesis-Associated Tissue Dysfunction

Aging is characterized by the progressive accumulation of somatic mutations. Acquisition of certain somatic mutations in hematopoietic stem cells (HSCs) confers on them a fitness advantage enabling them to outcompete other HSCs by clonal expansion in a phenomenon called clonal hematopoiesis (CH). CH is associated with a 40% increase in all-cause mortality linked to elevated risk for aging-associated morbidities such as cardiovascular disease (CVD), chronic kidney disease, COPD, and chronic liver disease. However, the mechanism by which CH exerts its adverse effects on disparate tissues is poorly understood. Here, we use CH-associated cardiac dysfunction as a paradigm to uncover general principles by which CH leads to the decline of tissue function. CH was modeled in mice by transplanting whole bone marrow (WBM) cells deficient for Tet2, a frequently mutated CH gene, into irradiated WT mice. We observed an elevated incidence of atrial fibrillation (AF) in Tet2 -/- WBM recipients relative to WT WBM recipients without any surgical or dietary intervention (Fig. A). The Tet2 -/- WBM recipients also had reduced left ventricular ejection fraction (LVEF), an indicator of heart failure. Mice exhibiting elevated incidence of AF and reduced LVEF are defined as having cardiac dysfunction. In addition, we observed that Tet2 -/- WBM recipients had increased systemic inflammation compared to controls, as evidenced by elevated levels of proinflammatory cytokines in the serum. Furthermore, their cardiac tissue exhibited upregulation of the inflammasome signaling pathway including the activation of Gasdermin D, the effector of pyroptotic cell death. Inflammation in the cardiac tissue was concomitantly associated with increased cardiac fibrosis in Tet2 -/- WBM recipients. Since inflammation is a known driver of CVD, we hypothesized that abrogating Gasdermin D-mediated inflammatory signaling could ameliorate CH-induced cardiac dysfunction. Indeed, Gsdmd -/- recipient mice were protected from cardiac dysfunction upon transplantation with Tet2 -/-WBM cells (Fig. A). Importantly, expansion of mutant hematopoietic cells was also mitigated in the absence of GSDMD: transplantation of Tet2 -/- WBM cells along with either WT or Gsdmd -/- WBM cells revealed that GSDMD deficiency restrained the rate of clonal expansion of Tet2 -/- cells (Fig. B). This suggests that instead of being mere bystanders, WT cells in the environment may play an active role in regulating the rate of mutant cell expansion via intercellular communication mediated by GSDMD. Identification of proinflammatory factors mediating clonal expansion and cardiac dysfunction is of significant interest for potential therapeutic intervention. Thus, we sought to determine factors that were upregulated in CH mouse models while being downregulated in Gsdmd -/- mice. Luminex multiplexed assay enabled us to identify several understudied proinflammatory cytokines present in serum satisfying the above criteria. In addition to elevated levels in the serum, some of these cytokines were upregulated in the cardiac tissue of Tet2 -/- WBM recipient mice but not in WT WBM recipients. Furthermore, some are reported to induce inflammasome signaling. CH, even in the absence of known drivers, is associated with higher CVD-associated mortality, suggesting the existence of pathways common to multiple forms of CH; systemic inflammation is one potential common mechanism. We tested whether aged WT WBM, which has low levels of mutant clone expansion but exhibits several features of CH, could cause cardiac dysfunction. We transplanted either aged or young WT WBM into young WT recipients and found that the aged WT WBM recipients exhibited elevated AF and reduced LVEF. These data suggest that aging-associated systemic inflammation may be sufficient for cardiac dysfunction. Importantly, one of the cytokines we uncovered was also upregulated in aged WT mice and has been associated with inflammation and fibrosis in multiple tissues. This cytokine is being further investigated as a top candidate for therapeutic intervention since it is a common feature of CH with and without identified drivers. Overall, our findings demonstrate the critical role of Gasdermin D-mediated inflammatory signaling in the pathogenesis of Tet2-mutant CH-induced cardiac dysfunction. We have also uncovered a cytokine that has emerged as a promising therapeutic target for CH-associated pathologies.

Dietary restriction mitigates the age-associated decline in mouse B cell receptor repertoire diversity

Aging impairs the capacity to respond to novel antigens, reducing immune protection against pathogens and vaccine efficacy. Dietary restriction (DR) extends life- and health span in diverse animals. However, little is known about the capacity of DR to combat the decline in immune function. Here, we study the changes in B cell receptor (BCR) repertoire during aging in DR and control mice. By sequencing the variable region of the BCR heavy chain in the spleen, we show that DR preserves diversity and attenuates the increase in clonal expansions throughout aging. Remarkably, mice starting DR in mid-life have repertoire diversity and clonal expansion rates indistinguishable from chronic DR mice. In contrast, in the intestine, these traits are unaffected by either age or DR. Reduced within-individual B cell repertoire diversity and increased clonal expansions are correlated with higher morbidity, suggesting a potential contribution of B cell repertoire dynamics to health during aging.

Halophytic Clonal Plant Species: Important Functional Aspects for Existence in Heterogeneous Saline Habitats.

Plant modularity-related traits are important ecological determinants of vegetation composition, dynamics, and resilience. While simple changes in plant biomass resulting from salt treatments are usually considered a sufficient indicator for resistance vs. susceptibility to salinity, plants with a clonal growth pattern show complex responses to changes in environmental conditions. Due to physiological integration, clonal plants often have adaptive advantages in highly heterogeneous or disturbed habitats. Although halophytes native to various heterogeneous habitats have been extensively studied, no special attention has been paid to the peculiarities of salt tolerance mechanisms of clonal halophytes. Therefore, the aim of the present review is to identify probable and possible halophytic plant species belonging to different types of clonal growth and to analyze available scientific information on responses to salinity in these species. Examples, including halophytes with different types of clonal growth, will be analyzed, such as based on differences in the degree of physiological integration, ramet persistence, rate of clonal expansion, salinity-induced clonality, etc.

Aberrant activation of TCL1A promotes stem cell expansion in clonal haematopoiesis.

Mutations in a diverse set of driver genes increase the fitness of haematopoietic stem cells (HSCs), leading to clonal haematopoiesis1. These lesions are precursors for blood cancers2-6, but the basis of their fitness advantage remains largely unknown, partly owing to a paucity of large cohorts in which the clonal expansion rate has been assessed by longitudinal sampling. Here, to circumvent this limitation, we developed a method to infer the expansion rate from data from a single time point. We applied this method to 5,071 people with clonal haematopoiesis. A genome-wide association study revealed that a common inherited polymorphism in the TCL1A promoter was associated with a slower expansion rate in clonal haematopoiesis overall, but the effect varied by driver gene. Those carrying this protective allele exhibited markedly reduced growth rates or prevalence of clones with driver mutations in TET2, ASXL1, SF3B1 and SRSF2, butthis effect was not seen inclones withdriver mutations in DNMT3A. TCL1A was not expressed in normal or DNMT3A-mutated HSCs, but the introduction of mutations in TET2 or ASXL1 led to the expression of TCL1A protein and the expansion of HSCs in vitro. The protective allele restricted TCL1A expression and expansion of mutant HSCs, as did experimentalknockdown of TCL1A expression. Forced expression of TCL1A promoted the expansion of human HSCs in vitro and mouse HSCs in vivo. Our results indicate that the fitness advantage of several commonly mutated driver genes in clonal haematopoiesis may be mediated by TCL1A activation.

Abstract 138: Establishment of monoclonal cell subpopulations as model systems for functionally exploring resistance mechanisms in EGFR mutant non-small cell cancers

Abstract Background: Coexisting subgroups of cells within a tumor are always evolving. Understanding the molecular events that drive this evolution remains a high priority for understanding dynamic response to treatment. However, models that accurately map individual clones’ activity and sensitivity to treatment remain a challenge. To fill this gap, we established monoclonal cell populations (MCP) from a cell line and used this model system to dissect coexisting cellular and molecular events within an individual tumor and modulated response to anti-EGFR treatment in Non-Small Cell Lung Cancers (NSCLC). Materials/Methods: MCPs were established from the commercially available H1975 EGFR-mutant NSCLC cell line. Parental cells were resuspended at a density of 10 cells per mL and 100 μL of cell suspension was added to each well of a 96-well plate. Eight plates were used, and each well was inspected 24 hours post-seeding by two or more independent scientists. Wells with individual cells were monitored every 3-4 days and followed throughout the expansion process. Once MCPs were established, morphological comparison was performed using the opensource QPath software and clones were classified as circular, branched, or mixed. Selected clones were then treated with 0.7 μM of Osimertinib for 72 hours, in line with the IC50 value of the parental line and treatment responses were compared. Results: Of the 120 single cells detected, 25 expanded successfully and 16 were treated with Osimertinib. Rates of clonal expansion varied from 19 to 49 days and the most prominent morphology was branched (68.75%), followed by mixed (18.75%) and circular (12.5%). No direct correlation was observed between rate of expansion and cell morphology. Response to Osimertinib varied significantly across the clones compared to the parental line from which they were established. Cell viability after 72 hours of treatment with Osimertinib was similar between the parental line and 5 of the 25 clones. For the remaining clones, cell viability was significantly lower in the monoclonal cell subpopulations compared to the parental line. Specifically, cell viability decreased by 40% compared to the parental cell in 5 clones. In the most sensitive clones, cell viability was reduced by 63% and 75% compared to the parental. Conclusion: 25 monoclonal cell subpopulations were successfully established and expanded from the H1975 cell line. MCPs showed significant variations in their growth rate and sensitivity to Osimertinib suggesting that this approach may help identify coexisting mechanisms of response to treatment within an individual tumor. Defining molecular profiles of coexisting MCPs may lead to the identification of biomarkers that may assist physicians in predicting response to treatment or design combination regimens that concomitantly target subclones within the tumor microecology. Citation Format: Claire Blanchard, Kyle Avery, Chelsea Ward, Abduljalil M. Alsubaie, Elisa Baldelli, Mariaelena Pierobon. Establishment of monoclonal cell subpopulations as model systems for functionally exploring resistance mechanisms in EGFR mutant non-small cell cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 138.

Life histories of myeloproliferative neoplasms inferred from phylogenies.

Mutations in cancer-associated genes drive tumour outgrowth, but our knowledge of the timing of driver mutations and subsequent clonal dynamics is limited1-3. Here, using whole-genome sequencing of 1,013 clonal haematopoietic colonies from 12 patients with myeloproliferative neoplasms, we identified 580,133 somatic mutations to reconstruct haematopoietic phylogenies and determine clonal histories. Driver mutations were estimated to occur early in life, including the in utero period. JAK2V617F was estimated to have been acquired by 33 weeks of gestation to 10.8 years of age in 5 patients in whom JAK2V617F was the first event. DNMT3A mutations were acquired by 8 weeks of gestation to 7.6 years of age in 4 patients, and a PPM1D mutation was acquired by 5.8 years of age. Additional genomic events occurred before or following JAK2V617F acquisition and as independent clonal expansions. Sequential driver mutation acquisition was separated by decades across life, often outcompeting ancestral clones. The mean latency between JAK2V617F acquisition and diagnosis was 30 years (range 11-54 years). Estimated historical rates of clonal expansion varied substantially (3% to 190% per year), increased with additional driver mutations, and predicted latency to diagnosis. Our study suggests that early driver mutation acquisition and life-long growth and evolution underlie adult myeloproliferative neoplasms, raising opportunities for earlier intervention and a new model for cancer development.

Clonal Hematopoiesis Is Driven By Aberrant Activation of TCL1A

Introduction: Clonal hematopoiesis of indeterminate potential (CHIP) may occur when a hematopoietic stem cell (HSC) acquires a fitness-increasing mutation resulting in its clonal expansion. A diverse set of driver genes, such as regulators of DNA methylation, splicing, and chromatin remodeling, have been associated with CHIP, but it remains largely unknown why HSCs bearing these mutations are positively selected. It has been challenging to identify the genetic and environmental factors mediating clonal expansion in humans, partially due to a lack of large cohorts with longitudinal blood sampling of participants. To circumvent this limitation, we developed a method to infer clonal expansion rate from single timepoint data called PACER (passenger-approximated clonal expansion rate).Methods: PACER is based on the principle that genomic passenger mutations can be used to infer the birth date of pre-malignant clones because these mutations accumulate fairly linearly over time. Thus, an individual with CHIP with a greater number of passenger mutations in the mutant clone is expected to have acquired the clone at a later age than someone with fewer passenger mutations. For two individuals of the same age and with clones of the same size, we expect the clone with more passengers to be more fit, as it expanded to the same size in less time. Typically, one would need to isolate single-cell colonies derived from HSCs in order to calculate the total passenger mutation burden. However, we hypothesized that this measure could also be approximated from whole genome sequencing of blood cell DNA, such as that used in large biobank projects. The expansion rate (PACER) is then estimated by adjusting the total passenger count for age and variant allele fraction in each individual. The ability of passengers to predict future clonal expansion was validated using longitudinal blood samples from 51 CHIP carriers in the Women's Health Initiative taken ~10 years apart (Figure 1). It also accurately predicted the known fitness effects due to different driver mutations in 5,551 CHIP carriers from the Trans-Omics for Precision Medicine (TOPMed) program (Figure 2).Results: Having validated the approach, we next hypothesized that we could identify germline variants influencing PACER, thus revealing genes and pathways mediating clonal expansion. The lead hit in a genome-wide association study (GWAS) of PACER was a common single nucleotide polymorphism (SNP) in the TCL1A promoter that was associated with slower clonal expansion in CHIP overall (Figure 3). TCL1A is an oncogene that is activated via translocation in T-cell prolymphocytic leukemia, but has no known role in CHIP or myeloid malignancies. A gene-level analysis indicated that the TCL1A SNP was associated with slower growth of clones bearing TET2 mutations, but had no effect on DNMT3A-mutant clone growth. We further found that those carrying two copies of the protective SNP had 40-80% reduced odds of having clones with driver mutations in TET2, ASXL1, SF3B1, SRSF2, and JAK2, but not DNMT3A. A concomitant decrease in incident myeloid malignancies was also seen in carriers of this protective SNP.Next, we interrogated how the protective SNP influenced TCL1A activity in HSCs. Normal human HSCs lacked open chromatin at the TCL1A promoter and TCL1A expression, but inducing frameshift mutations in TET2 via CRISPR editing led to accessibility of the promoter and gene/protein expression in HSCs (Figure 4). This effect was abrogated in HSCs from donors of the protective TCL1A SNP in a dose-dependent manner. Finally, we found that HSCs from donors homozygous for the protective SNP had markedly less expansion of phenotypic stem and progenitor cells in vitro after the introduction of TET2 mutations than TET2-edited HSCs from donors with two copies of the reference allele.Conclusions: In summary, we developed a novel method to infer the expansion rate of pre-malignant clones and performed the first ever GWAS for this trait. Our results indicate that the fitness advantage of several common driver genes in CHIP and hematological cancers is mediated through TCL1A activation, which may be a therapeutic target to treat these conditions. PACER is an approach that can be widely adopted to uncover genetic and environmental determinants of pre-malignant clonal expansion in blood and other tissues. [Display omitted] DisclosuresDesai: Bristol Myers Squibb: Consultancy; Kura Oncology: Consultancy; Agios: Consultancy; Astex: Research Funding; Takeda: Consultancy; Janssen R&D: Research Funding. Natarajan: Blackstone Life Sciences: Consultancy; Boston Scientific: Research Funding; Novartis: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding; Apple: Consultancy, Research Funding; Amgen: Research Funding; Genentech: Consultancy; Foresite Labs: Consultancy. Jaiswal: Novartis: Consultancy, Honoraria; AVRO Bio: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; Foresite Labs: Consultancy; Caylo: Current holder of stock options in a privately-held company.

Clonal Expansion of Tet2 +/- hematopoiesis Is Driven By Inflamm-Ageing Associated IL-1 Increase in Mice

Clonal Hematopoiesis of Indeterminate Potential (CHIP) is defined as the presence of an expanded somatic blood cell clone carrying a mutation in genes that are drivers of hematologic malignancy including DNMT3A, TET2, ASXL1, TP53, JAK2, and SF3B1, at a variant allele frequency (VAF) of at least 2% in the absence of other hematological abnormalities. CHIP has a prevalence of about 10% in the 70-80 year old population, further increases with ageing and associates with an increased risk of hematological malignancies, cardiovascular disease and all-cause mortality (Genovese et al. NEJM 2014; Jaiswal, S. et al. NEJM 2014). Recent studies indicate that higher pre-malignant clonal size and mutational burden increase the chances of malignant transformation in individuals carrying CHIP (Desai, P. Nat. Med. et al., 2018; Abelson, S. et al. Nature, 2018). While age is the best predictor of CHIP development and correlates directly with pre-malignant clonal size, the specific cellular-extrinsic factors promoting CHIP clonal expansion in the context of physiological aging are still unclear. We hypothesized that ageing associated low-grade inflammation (termed “inflamm-ageing”) is a driver of CHIP clonal expansion.We used standard bone marrow (BM) chimera models and developed a novel, irradiation independent, hematopoietic specific and tamoxifen inducible genetic mosaicism mouse model of Tet2 +/- driven CHIP (HSC-Scl-Cre-ERT; Tet2+/flox; R26 +/floxstop-EGFP triple transgenic mice) to determine the contribution of inflamm-aging factors to Tet2 +/- hematopoieticclonal expansion. Using these complementary models, we observe that peripheral Tet2 +/- clonal expansion rates increase with age (evident in erythroid, myeloid, lymphoid B and T lineages), which is paralleled by a significant expansion of Tet2 +/- hematopoietic stem and progenitor cell (HSPCs) populations in aged mice (12-14 months old). Importantly, Tet2 +/- clonal expansion associates with increased levels of inflammatory cytokine IL-1 in aged mice, which derives partially from Tet2 +/- mutant mature hematopoietic cells. To test the contribution of IL-1 to Tet2 +/- clonal expansion, we administered IL-1 (0.5ug/day for 14 days) to young CHIP carrying mice (2-4 months of age) and observed an IL-1R1-dependent expansion of Tet2 +/- hematopoietic mature lineages and HSPCs. Dissection of the cellular mechanisms operating downstream of IL-1/IL-1R1 revealed that Tet2 +/- clonal expansion results from increased multilineage differentiation and associates with increased HSPC cell-cycle progression (while not depending on IL-1-mediated effects on HSPC viability). Moreover, Tet2 +/- HSPCs show a higher in vitro and in vivo repopulation capacity in response to prolonged IL-1 exposure compared to their WT counterparts. Finally, to directly test the contribution of IL-1 to drive Tet2 +/- clonal expansion in the context of physiological aging, we set up genetic (BM chimeras using donor BM from Tet2 +/-; Il-1r1-/- compound mutants) or pharmacological inhibited IL-1 signaling (Anakinra, hIL-1ra) during mouse ageing. Strikingly, both approaches prevented ageing-dependent Tet2 +/- clonal expansion, thus confirming IL-1 as key “inflamm-ageing” driver of Tet2 +/- clonal expansion. Overall, our data provide proof-of-concept that IL-1 production derived from aged BM cells is a relevant and targetable driver of Tet2 +/- clonal expansion in aged mice. DisclosuresManz: CDR-Life Inc: Consultancy, Current holder of stock options in a privately-held company; University of Zurich: Patents & Royalties: CD117xCD3 TEA.

Par-4 Overexpression Impedes Leukemogenesis in the Eµ-TCL1 Leukemia Model

Introduction: Prostate apoptosis response 4 (Par-4) is a tumor suppressor protein that is commonly downregulated by promoter methylation, inactivated by Akt1, or mutated in diverse solid tumors. It exhibits a unique cancer-specific activation quality, sensitizing tumor cells to apoptosis and inhibiting proliferation, but not affecting the untransformed cell type of origin. This is thought to be due to hyperactivation of kinases that activate Par-4 in transformed cells, which results from dysregulation of signaling pathways during transformation. In chronic lymphocytic leukemia (CLL) Par-4 is not downregulated, but is in fact more abundant than in healthy B cells. We explored the therapeutic potential of targeting the Par-4 pathway in CLL to exploit an endogenous anti-tumor mechanism.Methods: The Eµ-TCL1 mouse is a leukemia model commonly used to study CLL biology and test experimental therapeutics. We have developed the first B cell specific human Par-4 (Eµ-Par-4) overexpression mouse to explore the effect of Par-4 overexpression in the TCL1 model. Double transgenic Par-4/TCL1 mice were bred from single transgenic mice to produce a B cell specific Par-4 overexpression leukemia model. TCL1 CLL-like B cells express CD5 and CD19, and are abundant in the peripheral blood, bone marrow and spleen. Monthly bleeding for peripheral blood counts and spleen palpation were used to track disease progression in these mice, with the tumor burden in the peripheral blood being roughly equivalent to the severity of disease in this model. Mice with advanced disease show vastly increased white blood cell (WBC) counts, with high proportions of WBCs being the characteristic CD5 positive leukemic B cell, splenomegaly, and various cytopenias.Results: Disease burden in single transgenic TCL1 mice, estimated by abundance of CD45+/CD5+/CD19+ B cells in the peripheral blood, was found to be significantly higher than in the Par-4/TCL1 mice bred from the same founder at 10 months of age (p=.0099). The percentage of CD5+/CD19+ WBCs in the peripheral blood of TCL1 mice averaged 74% while the Par-4/TCL1 mice averaged 37%. TCL1 white blood cell counts were also found to be significantly higher than syngeneic Par-4/TCL1 mice (p=.0028) at 10 months, averaging 65000/µL compared to just 15000/µL in the double transgenic. Animals that met euthanasia criteria were analyzed histologically for infiltration of the spleen and bone marrow by malignant B cells. The degree of CLL infiltration was similar in both the TCL1 single transgenics and the Par-4/TCL1 double transgenics, indicating that while the disease progression is stifled, the cancer does adapt and resume course with similar pathology. EdU incorporation assays were performed to estimate relative rates of clonal expansion in the splenic B cell population. Expansion of CD5+/CD19+ splenocytes was found to be higher in the TCL1 single transgenic mice than the Par-4/TCL1 transgenics (p=.01). We believe this suggests a cell intrinsic defect, imposed by overexpression of Par-4 protein, which creates a hurdle that must be overcome for the malignancy to progress. Mechanistic studies indicate that Par-4 mediated inhibition of NF-κB signaling in leukemic B cells may be responsible for the lag in disease progression in Par-4/TCL1 mice. Immunoblotting revealed that IκBα levels are higher and the proportion of phosphorylated protein is lower in Par-4/TLC1 splenic B cells, relative to single transgenic TCL1 B cells.Conclusions: We have shown that human Par-4 overexpression in a murine leukemia model is capable of influencing disease progression by slowing expansion of malignant cells. Inhibition of the NF-κB pathway by stabilization of IkBα appears to be the mechanism of action, which would place Par-4 in the canonical NF-κB pathway. We know this pathway is integral to CLL cell survival and is downregulated by effective therapeutic agents. These findings indicate that the Par-4 is capable of suppressing development of TCL1 driven leukemia by downregulating survival signals that are essential to apoptosis evasion and proliferation in CLL. These studies warrant further characterization of Par-4 pathway in human CLL cells. DisclosuresByrd:Janssen: Research Funding; Pharmacyclics: Research Funding; Genentech: Research Funding; The Ohio State University: Patents & Royalties: OSU-2S; Acerta Pharma: Research Funding.

Driver Mutation Acquisition in Utero and Childhood Followed By Lifelong Clonal Evolution Underlie Myeloproliferative Neoplasms

Background Recurrent mutations in cancer-associated genes drive tumour outgrowth, however, the timing of driver mutations and the dynamics of clonal expansion remain largely unknown. Philadelphia-negative myeloproliferative neoplasms (MPN) are unique cancers capturing the earliest stages of tumorigenesis through to disease evolution. Most patients harbor JAK2V617F, present as the only driver mutation or occurring in combination with driver mutations in genes such as DNMT3A or TET2. We aimed to identify the timing of driver mutations and clonal dynamics in adult MPN. Method We undertook whole-genome sequencing of individual single-cell derived hematopoietic colonies (n=952) together with targeted resequencing of longitudinal blood samples from 10 patients with MPN who presented with disease between ages 20 and 76 years. We identified 448,553 somatic mutations which were used to reconstruct phylogenetic trees of hematopoiesis, tracing blood cell lineages back to embryogenesis. We timed driver mutation acquisition, characterised the dynamics of tumour evolution and measured clonal expansion rates over the lifetime of patients. Resequencing of bulk blood samples corroborated clonal trajectories and provided population estimates. Results JAK2V617F was acquired in utero or childhood in all patients in whom JAK2V617F was the first or the only driver mutation. Earliest age estimates were within a few weeks post conception, and upper estimates of age of acquisition were between 4.1 months and 11.4 years, despite wide ranging ages of MPN presentation. The mean latency between JAK2V617F acquisition and clinical presentation was 34 years (range 20-54 years). Subsequent driver mutation acquisition, including for JAK2V617F, was separated by decades. Disease latency following acquisition of JAK2V617F as a second driver event was still 12-27 years. DNMT3A mutations, commonly associated with age-related clonal hematopoiesis (CH), occurred as the first driver event, subsequent to mutated-JAK2, and as independent clones representing CH in MPN patients. DNMT3A mutations were also first acquired in utero or childhood, at the earliest 1.2 weeks post conception, and the latest 7.9 weeks of gestation to 7.8 years across 4 patients. A recurrent feature of the clonal landscape in MPN was the observation of similar genetic changes repeatedly occurring in unrelated clones within the same patient. Such 'parallel evolution' was observed for chr9p loss-of-heterozygosity, chr1q+ and mutations in myeloid cancer genes, suggesting that patient-specific factors flavour selective landscapes in MPN. Normal hematopoietic stem cells accumulated ~18 somatic mutations/year, however, mutant clones, particularly those with mutant-JAK2, acquired 1.5-5.5 excess mutations/ year and had shorter telomeres, reflecting increased cell divisions during clonal expansion. We modelled the rates of clonal expansion and found that they varied substantially, both across patients and within individuals. In one patient, an in utero acquired DNMT3A-mutated clone expanded slowly at <10%/year, taking 30 years to reach a clonal fraction of 1%, whilst a clone with mutated-JAK2, -DNMT3A and -TET2 expanded at >200%/year, doubling in size every 7 months. JAK2V617F as a single driver mutation also expanded variably across patients, highlighting that other factors, which may include germline, cytokine or stem cell differences between individuals, also influence selection for driver mutations. JAK2V617F associated clonal expansion rates in MPN were greater than that reported for JAK2-CH. Furthermore, rates of expansion in the cohort predicted time to clinical presentation, more so than age of mutation acquisition or tumour burden at diagnosis. This suggests that JAK2-mutant clonal expansion rates determine both if and when clinical manifestations occur. Driver mutations and rates of clonal expansion would have been detectable in blood one to four decades before clinical presentation. Conclusions MPN originate from driver mutation acquisition very early in life, even before birth, with life-long clonal expansion and evolution, establishing a new paradigm for blood cancer development. Early detection of mutant-JAK2 together with determination of clonal expansion rates could provide opportunities for early interventions aimed at minimising thrombotic risk and targeting the mutant clone in at risk individuals. Disclosures No relevant conflicts of interest to declare.

Different Mechanisms of Cigarette Smoking-Induced Lung Cancer.

The risk of cigarette smoking plays a pivotal role in increasing the incidence rates of lung cancer. This paper sheds new light on modeling the impact of cigarette smoking on lung cancer evolution, especially genetic instability and the number of gene mutations in the genome of stem cells. To handle this issue, we have set up stochastic multi-stage models to fit the data set of the probabilities of current and former smokers from the Nurses' Health Study cohort of females (NHS) and the Health Professionals Follow up Study cohort of men (HPFS). Throughout this paper, we consider both mutation rates and clonal expansion rates as parameters in each compartment. For current and former smokers, three-driver mutations are most likely to take place in the progression of lung cancer under smoking risk. For current smokers, our findings reveal that two to sixteen gene mutations are required to obtain a cancerous cell among men and women in US. Moreover, two to six (eleven) cancer-mutations are available in the pathway to lung cancer among former smokers who have quit smoking for more (less) than ten years for both male and female patients. This highlights that cigarette smoking stimulates the number of driver mutations during lung tumorigenesis in both sexes. It is very crucial to examine the role of cigarette smoking in determining whether genomic instability is an early stage or late stage in the process of lung carcinogenesis.

Mathematical modeling of female breast cancer in Japan

Cancer incidence rates are significantly different all over the world. Breast cancer is affected by many factors, the most important being genetics and lifestyle. The aim of this paper is to study the mutation mechanisms of breast cancer for Japanese women by fitting the incidence data of three high-quality population areas in Japan from 1985 to 2010. To achieve this goal, we have set up multi-stage models within the mathematical model of Moolgavkar, Venzon, and Knudson. Such models take both mutation rates and clonal expansion rates as parameters in each compartment into consideration. Based on our simulation outcomes, two to twelve driver mutations are sufficient in the pathway to female breast cancer in Japan. On the other hand, a previous study demonstrated that breast cancer in American women requires two to fourteen gene mutations to get a cancerous cell. Moreover, the 3-stage mathematical model is the optimal model as it fits clinical data very nicely for all affected cases of females in Japan and the US. The genetic instability has a prominent effect on the tumorigenesis of Japanese females caused by the first four driver mutations. The calculated results for Japanese women are compared with previous works for American women.

QUANTITATIVE ANALYSIS OF THE POTENTIAL ROLE OF BASAL CELL HYPERPLASIA IN THE RELATIONSHIP BETWEEN CLONAL EXPANSION AND RADON CONCENTRATION.

Applying the two-stage clonal expansion model to epidemiology of lung cancer among uranium miners, it has been revealed that radon acts as a promoting agent facilitating the clonal expansion of already mutated cells. Clonal expansion rate increases non-linearly by radon concentration showing a plateau above a given exposure rate. The underlying mechanisms remain unclear. Earlier we proposed that progenitor cell hyperplasia may be induced upon chronic radon exposure. The objective of the present study is to test whether the induction of hyperplasia may provide a quantitative explanation for the plateau in clonal expansion rate. For this purpose, computational epithelium models were prepared with different number of basal cells. Cell nucleus hits were computed by an own-developed Monte-Carlo code. Surviving fractions were estimated based on the number of cell nucleus hits. Cell division rate was computed supposing equilibrium between cell death and cell division. It was also supposed that clonal expansion rate is proportional to cell division rate, and therefore the relative increase in cell division rate and clonal expansion rate are the same functions of exposure rate. While the simulation results highly depend on model parameters with high uncertainty, a parameter set has been found resulting in a cell division rate-exposure rate relationship corresponding to the plateau in clonal expansion rate. Due to the high uncertainty of the applied parameters, however, further studies are required to decide whether the induction of hyperplasia is responsible for the non-linear increase in clonal expansion rate or not. Nevertheless, the present study exemplifies how computational modelling can contribute to the integration of observational and experimental radiation protection research.

The effect of cigarette smoking on lung cancer evolution.

The aim of this paper is to elucidate the risk of lung carcinogenesis from cigarette smoking among current and former smokers. To achieve this goal, we have set up a stochastic three-stage model to fit the data of Surveillance, Epidemiology, and End Results (SEER) program besides the data set of smoking derived from the Nurses' Health Study cohort of females (NHS) and the Health Professionals Follow up Study cohort of men (HPFS). The calculations are performed by considering both mutation and clonal expansion rates as parameters in each compartment. For current smokers, our findings show that cigarette smoking has more significant impact on the mutation rates of cells than the clonal expansion rates of premalignant cells among men and women. In particular, for male patients, cigarette smoking affects the mutation in normal cells and the transformation from premalignant cells to malignant ones in the optimal model. In addition, cigarette smoking induces only the initial mutation rates in normal cells among American women. For current and former smokers, cigarette smoking stimulates only the clonal expansion rate of the first premalignant cells in both sexes. However, we find that the impact of cigarette smoking is minimal in former smokers who have stopped smoking for over ten years among men and women in US.